[PDF] Study Of Non Covalent Multisubunit Protein Carbohydrate Interactions By Electrospray Ionization Mass Spectrometry eBook
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This thesis describes the development and application of electrospray ionization mass spectrometry (ESI-MS) based techniques to investigate protein-carbohydrate interactions in vitro. A catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay was developed for the identification of specific interactions between water-soluble multisubunit proteins and glycosphingolipids (GSL). The assay is of high sensitivity and specificity, and demonstrates the potential for discovering biologically relevant protein-GSL interactions. Collision-induced dissociation (CID) experiments and molecular dynamic simulations were performed to investigate the dissociation pathways of multisubunit protein-ligand complexes in the gas phase. The observation of multiple dissociation pathways suggests that collisional activation of multisubunit protein-ligand complexes in the gas phase is likely to induce significant changes to the nature of the protein-ligand interactions.
The interactions between water-soluble proteins and carbohydrates found on the surfaces of cells play important roles in many physiological and pathological cellular processes. Carbohydrates function as receptors for signaling, cellular recognition and adhesion, and pathogen infections. This thesis focuses on the development and application of electrospray ionization mass spectrometry (ESI-MS) methods to detect and quantify protein-carbohydrate interactions in vitro. In Chapter 2, the intrinsic affinities (per binding site) of the protruding domain dimer (P dimer, 69 kDa) of the human norovirus (NoV) strain VA387 to a panel of 47 soluble analogs of histo-blood group antigens (HBGAs) were quantified using the direct ESI-MS assay. Our results revealed that the P dimer exhibits a broad specificity for the HBGAs and bind, albeit weakly (intrinsic association constants (Ka,int) of 102 - 103 M-1), to all of the oligosaccharides tested. Overall, the A and B antigens exhibit stronger binding than the H and Lewis antigens. In addition, the affinities are also affected by the precursor chain type of HBGAs but not by the chain length. In Chapter 3, the applicability of the catch-and-release (CaR)-ESI-MS assay for screening carbohydrate libraries against large protein complexes was demonstrated for the first time. Libraries containing as many as 146 compounds were screened against NoV VA387 subviral P particle (24-mer, 865 kDa). Notably, the results of the screening experiments revealed NoV interactions with oligosaccharides with structures found in human milk and the cell wall of mycobacteria. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors. In Chapters 4 and 6, the direct ESI-MS assay was combined with a competitive binding strategy in order to measure the affinities of protein-carbohydrate interactions that can't be directly quantified by ESI-MS. In Chapter 4, the affinities of the NoV P particle and virus-like particle (VLP, 180-mer, 10.5 MDa) to HBGA ligands were quantified using the proxy protein ESI-MS method, which utilizes competitive protein binding. The results revealed that HBGA ligands exhibit similar affinities for the P particle and P dimer whereas the HBGA affinities for the VLP are consistently higher than those measured for the P dimer, but within a factor of three. In Chapter 6, the proxy ligand ESI-MS assay, which is on the basis of competitive ligand binding, combined with nanodisc technology to solubilize glycolipids was used to determine the interactions of cholera toxin B subunit homopentamer (CTB5) with GM1, and a family 51 carbohydrate-binding module (CBM) with B type 2 tetrasaccharide neoglycolipid. A notable finding of this study is that the affinities of the glycolipid ligands in the nanodisc are lower, by a factor of ≤5, than those of the corresponding oligosaccharides in solution. In Chapter 5, the screening using CaR-ESI-MS assay revealed the first evidence that human NoVs bind to gangliosides. Moreover, affinities measurements were reported for the NoV VA387 P dimer, P particle and VLP, and VA115 P dimer for a series of ganglioside oligosaccharides. Notably, the ganglioside affinities are similar in magnitude to those of HBGA receptors for NoVs. Additional confirmation of NoV-ganglioside interactions was provided by the binding measurements using the enzyme-linked immunosorbent assays. Finally, also in Chapter 6, a systematic ESI-MS investigation aimed at elucidating the processes that influence binding of water-soluble proteins to glycolipids incorporated into nanodiscs was described. The interactions of CTB5 to GM1 nanodiscs studied by ESI-MS indicated that proteins bind reversibly to nanodisc-associated glycolipids, and that proteins possessing multiple ligand binding sites are able to interact with glycolipids originating from different nanodiscs. Moreover, the nature of the protein-glycolipid complexes detected by ESI-MS is likely to be influenced by the diffusion of glycolipids between nanodiscs.
The authoritative guide to analyzing protein interactions by mass spectrometry Mass spectrometry (MS) is playing an increasingly important role in the study of protein interactions. Mass Spectrometry of Protein Interactionspresents timely and definitive discussions of the diverse range of approaches for studying protein interactions by mass spectrometry with an extensive set of references to the primary literature. Each chapter is written by authors or teams of authors who are international authorities in their fields. This leading reference text: * Discusses the direct detection of protein interactions through electrospray ionization (ESI-MS); ion mobility analysis; and matrix-assisted laser desorption/ionization (MALDI-MS) * Covers the indirect analysis of protein interactions through hydrogen-deuterium exchange (HX-MS); limited proteolysis; cross-linking; and radial probe (RP-MS) * Guides researchers in the use of mass spectrometry in structural biology, biochemistry, and protein science to map and define the huge number and diversity of protein interactions * Reviews the latest discoveries and applications and addresses new and ongoing challenges This is a comprehensive reference for researchers in academia and industry engaged in studies of protein interactions and an excellent text for graduate and postgraduate students.